Mitochondrial Dysfunction Induced by Xenobiotics: Involvement in Steatosis and Steatohepatitis

C H A P T E R

15 Mitochondrial Dysfunction Induced by Xenobiotics: Involvement in Steatosis and Steatohepatitis Karima Begriche*, Julie Massart†, Bernard Fromenty* ⁎

INSERM, Univ Rennes, INRA, Institut NUMECAN (Nutrition Metabolisms and Cancer) UMR_A 1341, UMR_S 1241, Rennes, France †Department of Molecular Medicine and Surgery, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden

1 INTRODUCTION Steatosis, also referred to as fatty liver, is a hepatic lesion that can be frequently induced by ethanol overconsumption and many xenobiotics, such as drugs and environmental pollutants.1–3 Notably, fat accumulation in the liver (mainly as triglycerides) is also present in a majority of obese individuals, even in those who are not drinking alcohol or are unexposed to any steatogenic xenobiotics. Although fatty liver itself is benign, it can progress in the long term to steatohepatitis, which is characterized by necroinflammation and fibrosis.2, 4 The occurrence of steatohepatitis is a major issue for patients and physicians because the lesion can evolve toward cirrhosis and hepatocellular carcinoma (HCC). The large spectrum of liver lesions that can occur in obese individuals is referred to as nonalcoholic fatty liver disease (NAFLD). Some clinical and experimental investigations reported that some xenobiotics can worsen NAFLD.5, 6 It has been acknowledged that mitochondrial dysfunction plays a major role in the pathogenesis of steatosis and steatohepatitis induced by numerous xenobiotics. In NAFLD, mitochondrial dysfunction does not seem to play a central role in fat deposition but rather appears to have a significant pathogenic role in the progression of fatty liver to nonalcoholic steatohepatitis (NASH).7, 8 More specifically, the upregulation of the mitochondrial oxidation of different substrates and the concomitant impairment of the mitochondrial respiratory chain (MRC) activity favor the overproduction of reactive oxygen species (ROS), which seems to play a pivotal role in necroinflammation and fibrosis.7, 9 In this chapter, we examine how xenobiotics can induce steatosis and steatohepatitis secondary to mitochondrial dysfunction. We selected xenobiotics able to alter mitochondrial function by different mechanisms, such as

Mitochondria in Obesity and Type 2 Diabetes https://doi.org/10.1016/B978-0-12-811752-1.00015-8

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Examples of toxic compounds NRTIs, ethanol, B[a]P, CCl4

Mitochondrial toxicity

Hepatic and metabolic consequences

mtDNA depletion Decreased synthesis of 13 MRC proteins

Amiodarone, tamoxifen, ethanol, B[a]P, CCl4

Inhibition of MRC

Reduced ATP, cytolysis, ROS overproduction, lactic acidosis

Reduced NADH and FADH2 oxidation

Amiodarone, tamoxifen, ethanol

mtFAO impairment

Steatosis, reduced ATP, cytolysis

FIG.  1  Toxic compounds can impair mitochondrial function in the liver via different mechanisms, including mtDNA depletion, inhibition of MRC, and impairment of the mtFAO pathway. Ethanol overconsumption and some xenobiotics, such as amiodarone and CCl4, can impair mitochondrial function by two or three different mechanisms. The main hepatic and metabolic consequences of these mitochondrial effects are indicated in the right part of the figure. Impairment of MRC and/or mtFAO can reduce liver ATP synthesis. MRC inhibition will lead to mtFAO impairment only if MRC activity is reduced severely. Depending on the severity of ATP depletion, the spectrum of hepatic cytolysis can vary from isolated increase in plasma ALT and AST to fulminant hepatitis and severe hepatic dysfunction. Impairment of mtFAO eventually leads to steatosis, corresponding to an accumulation of lipids (mainly triglycerides). Inhibition of MRC also can favor the overproduction of mitochondrial ROS, which play a major role in the progression of steatosis to steatohepatitis. Lactic acidosis, which results from tricarboxylic acid cycle impairment, is observed mainly with NRTIs such as stavudine and didanosine. Further information can be found in the text. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; B[a]P, benzo[a]pyrene; CCl4, carbon tetrachloride; MRC, mitochondrial respiratory chain; mtDNA, mitochondrial DNA; mtFAO, mitochondrial fatty acid oxidation; NRTIs, nucleoside reverse transcriptase inhibitors; ROS, reactive oxygen species.

direct inhibition of mitochondrial fatty acid oxidation (mtFAO), impairment of mitochondrial DNA (mtDNA) replication, and oxidative stress (Fig. 1). When data are available, we indicate whether these xenobiotics were shown (or suspected) to worsen NAFLD related to obesity. Other examples of xenobiotics inducing steatosis and steatohepatitis via mitochondrial dysfunction can be found in several recent reviews.1, 2, 10–13

2  THE MAIN FEATURES OF MITOCHONDRIA The first chapters of this book provide exhaustive information concerning mitochondrial function and structure. Therefore, the paragraphs below just give the essential information to comprehend how xenobiotics can induce mitochondrial dysfunction and the main metabolic consequences of such effect, in particular regarding fat accumulation and ROS overproduction.

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2.1  The Oxidative Phosphorylation Process Mitochondria provide most of the ATP required for cell function and survival. ATP synthesis is performed via the oxidative phosphorylation (OXPHOS) process, which couples the oxidation of different substrates to ATP generation.14, 15 The constant oxidation of substrates such as fatty acids and pyruvate generates FADH2 and NADH that subsequently transfer their electrons to the mitochondrial respiratory chain (MRC), thus regenerating the NAD+ and FAD necessary for other cycles of fuel oxidation. The electrons provided by NADH or FADH2 migrate along the respiratory chain, up to cytochrome c oxidase (COX), where they safely react with oxygen and protons to form water. Electron transfer across MRC complexes I, III, and IV is coupled with the extrusion of protons from the mitochondrial matrix into the intermembrane space of mitochondria, creating a large electrochemical potential (Δψ) across the inner membrane. When ADP is high, protons reenter the matrix through the F0 portion of ATP synthase, causing the conversion of ADP into ATP by the F1 portion of this enzyme. OXPHOS can be uncoupled by different xenobiotics including drugs, as discussed below.

2.2  The Mitochondrial Production of ROS MRC is deemed to be the main site of ROS production in most cells. A small fraction of electrons going through the MRC complexes I and III constantly leaks from these complexes and react with oxygen to form the superoxide anion radical (O2-.).7, 14 This radical, however, is mostly dismutated by manganese superoxide dismutase (MnSOD, also referred to as SOD2) into hydrogen peroxide (H2O2), which is more stable compared to the superoxide anion radical. Mitochondrial H2O2 can be subsequently detoxified into water by peroxiredoxins and glutathione peroxidases (GPXs), which necessitate reduced glutathione (GSH) as cofactor.14 Therefore, the mitochondrial GSH store plays a major role by avoiding the excessive accumulation of hydrogen peroxide and subsequent oxidative stress.16 Whereas mitochondrial ROS serve as key signaling molecules when produced at physiological levels,17, 18 ROS overproduction secondary to mitochondrial dysfunction can have different deleterious effects in the liver.

2.3  Oxidation of Fatty Acids Fatty acids (FAs) of different lengths can be oxidized by the mtFAO pathway, a key metabolic process mandatory for the preservation of normal energy output, especially during fasting.19 Whereas short-chain FAs (SCFAs) and medium-chain FAs (MCFAs) freely enter mitochondria, the entry of long-chain FAs (LCFAs) into mitochondria requires carnitine palmitoyltransferase 1 (CPT1) and 2 (CPT2). During their β-oxidation, SCFAs, MCFAs, and LCFAs undergo four sequential reactions leading to the release of one acetyl-CoA molecule and a shortened FA, which can be further oxidized by other cycles of mtFAO. Two of these reactions are catalyzed by different FAD- and NAD+-dependent dehydrogenases that have specific activities for SCFAs, MCFAs, or LCFAs.19 In the liver, a significant part of the mtFAO-derived acetyl-CoA molecules generate the ketone bodies acetoacetate and β-hydroxybutyrate, especially during fasting. Importantly, some enzymes of the mitochondria β-oxidation pathway can produce a significant amount of hydrogen peroxide.20, 21

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2.4  The Mitochondrial Genome Mitochondria contain their own genome, as well as all the components (e.g., enzymes and transcription factors) required for its replication, transcription, translation, and repair. mtDNA is a 16.6 kb double-strand circular genome present within each mitochondrion in several copies and encodes 13 MRC polypeptides. These polypeptides are then inserted in the inner membrane within the MRC complexes I, III, IV (cytochrome c oxidase), and V (ATP synthase), along with dozens of nuclear DNA-encoded proteins. Permanent mtDNA replication by the DNA polymerase γ keeps levels of cellular mtDNA constant in spite of continuous degradation of the most damaged and/or dysfunctional mitochondria. mtDNA seems to be more prone to oxidative damage than nuclear DNA, which eventually can lead to the occurrence of point mutations and deletions.22 Oxidative damage to mtDNA can also induce a strong reduction of the mtDNA copy number and subsequent OXPHOS deficiency.22, 23

3  MAIN CONSEQUENCES OF MRC AND mtFAO INHIBITION MRC inhibition can induce significant ATP depletion, leading to cell death by necrosis.24, 25 In the liver, ATP shortage can induce cytolytic hepatitis (also called hepatic cytolysis) that comprises a large spectrum of liver injuries of different severity (Fig. 1). Although the mildest forms of hepatic cytolysis are characterized by an isolated increase in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the most severe cases can be associated with fulminant hepatitis and severe hepatic dysfunction. Moreover, hepatic cytolysis can favor inflammation through the release of endogenous damage-associated molecular patterns (DAMPS).26 Mild hepatic cytolysis accompanied by inflammation are key features of steatohepatitis, whatever its etiology.27–29 Another important consequence of MRC inhibition is the secondary impairment of mtFAO and tricarboxylic acid (TCA) cycle (Fig.  1). Lower oxidation of NADH and FADH2 strongly reduces FAD and NAD+ levels, thus hindering the activity of the different FAD- and NAD+dependent dehydrogenases of these metabolic pathways.2, 27 MRC inhibition, however, will secondarily lead to an impairment of mtFAO and TCA cycle only if MRC activity is reduced severely.2, 25, 27 Inhibition of mtFAO can lead to lipid accumulation (Fig. 1) as discussed below. Impairment of the TCA cycle can induce hyperlactatemia and lactic acidosis because the conversion of unmetabolized pyruvate to lactate by lactate dehydrogenase is favored by NADH accumulation.30, 31 Finally, a significant inhibition of MRC can also favor ROS overproduction, in particular at the level of complexes I and III.24, 25, 32 In the liver, overproduction of mitochondrial ROS induced by xenobiotics can favor oxidative stress, inflammation, and fibrosis.24, 30, 33 Therefore, mitochondrial ROS overproduction appears to play a key role in the progression of simple steatosis toward steatohepatitis. This key pathophysiological feature seems to be applicable for different causes of steatosis such as drugs, ethanol, different types of toxins, and obesity, or any combination of these factors (Fig. 2).6, 7, 12 Inhibition of mtFAO can lead to hepatic cytolysis (Fig.  1), because this metabolic pathway provides most of the ATP required for cell homeostasis and function, especially during fasting.25, 27 Xenobiotic-induced inhibition of mtFAO also can induce steatosis (Fig. 1), or can aggravate preexisting fatty liver linked to obesity (Fig.  2).2, 6 Although most of the stored

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4 Drugs

Xenobiotics

Inhibition of MRC

 mtFAO

 ROS

Aggravation of fatty liver

 ATP

NASH

FIG.  2  Xenobiotic-induced mitochondrial dysfunction can aggravate fatty liver and promote its progression to NASH in obese patients. Some xenobiotics are able to impair the mtFAO pathway either directly or secondary to MRC inhibition, thus aggravating lipid accumulation in the fatty liver. In case of MRC inhibition, a faster progression of fatty liver to NASH can occur via a reduction of ATP synthesis and higher mitochondrial ROS production. These effects can promote hepatic cytolysis, inflammation, and fibrosis in the context of fatty liver. In addition to mitochondrial dysfunction, some xenobiotics can also aggravate fatty liver by favoring de novo lipogenesis and/or reducing VLDL secretion. Further information can be found in the text. Abbreviations: MRC, mitochondrial respiratory chain; mtFAO, mitochondrial fatty acid oxidation; NASH, nonalcoholic steatohepatitis; ROS, reactive oxygen species; VLDL, very-low density lipoprotein.

lipids are triglycerides, free fatty acids and dicarboxylic acids can also accumulate in a significant amount.27, 34 This could reinforce drug-induced mitochondrial dysfunction because these lipid derivatives are able to impair OXPHOS and TCA cycle, or increase the permeability of the mitochondrial membranes.25, 27, 35 A severe inhibition of mtFAO can also alter the hepatic gluconeogenesis pathway, leading to hypoglycemia.27 The strong reduction in acetyl-CoA levels impairs the activity of pyruvate carboxylase, a major regulatory enzyme in the gluconeogenesis process.25, 27

4 DRUGS 4.1 Amiodarone Amiodarone is a broad-spectrum antiarrhythmic drug that also presents an antianginal effect. Amiodarone-induced hepatotoxicity includes acute hepatic cytolysis, cholestasis, steatosis, steatohepatitis, and cirrhosis.27, 36 Numerous studies have shown that mitochondrial dysfunction is a major mechanism whereby amiodarone can induce steatosis and steatohepatitis.12, 25, 27 Investigations performed on isolated rodent mitochondria revealed that amiodarone presents a dual effect on mitochondrial respiration and OXPHOS, depending on its concentration.37–40 At low concentrations (20–100 μM), amiodarone uncouples OXPHOS and stimulates mitochondrial respiration via a protonophoric effect. This cationic amphiphilic molecule can be protonated within the mitochondrial intermembrane space and electrophoretically transported into the mitochondrial matrix by using the membrane potential ΔΨ. At higher concentrations (>100 μM), however, the progressive intramitochondrial accumulation

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of amiodarone rapidly inhibits MRC activity. Notably, both OXPHOS uncoupling and MRC inhibition can reduce ATP synthesis so that amiodarone-induced energy shortage can occur even at low intracellular concentrations of this drug.40, 41 Amiodarone-induced MRC inhibition is most probably the main mechanism whereby this drug induces mitochondrial ROS overproduction, oxidative stress, and lipid peroxidation (Fig. 2).39, 42, 43 Amiodarone could also favor oxidative stress via GSH depletion because of the formation of GSH conjugates with the drug and its diquinone metabolites44 and via a reduction of GPX expression.43 In vitro and in vivo investigations also reported that amiodarone is able to inhibit mtFAO.38, 40, 45 Although this effect could be secondary to MRC inhibition, some experiments suggested that mtFAO could be directly inhibited by amiodarone (Fig. 2), in particular at the level of long-chain acyl-CoA dehydrogenase (LCAD) and CPT1.38, 39, 46, 47 Lastly, perhexiline and 4,4′-diethylaminoethoxyhexestrol (DEAEH), two cationic amphiphilic drugs withdrawn from the market because of frequent steatohepatitis, were shown to alter mitochondrial function in a similar manner to amiodarone.42, 47, 48

4.2  Nucleoside Reverse Transcriptase Inhibitors Nucleoside reverse transcriptase inhibitors (NRTIs) are the first antiretroviral drugs marketed for the treatment of human immunodeficiency virus (HIV) infection. This pharmacological class includes zidovudine (AZT), stavudine (d4T), didanosine (ddI), and zalcitabine (ddC). NRTIs are 2′,3′-dideoxynucleoside analogues in which the 3′-hydroxyl group of the sugar ring is replaced by either a hydrogen atom or another group unable to form a phosphodiester linkage. The lack of this 3′-hydroxyl group is required for the inhibition of HIVreverse transcriptase activity. NRTI-induced liver injury includes hepatic cytolysis, microvesicular and/or macrovacuolar steatosis, steatohepatitis, cirrhosis, and cholestasis.2, 36 Numerous clinical and experimental investigations showed that NRTI-induced hepatotoxicity and other adverse effects are the consequence of an impairment of mtDNA replication.22, 27, 30, 49, 50 By impairing mtDNA replication, these drugs can induce severe mtDNA depletion and OXPHOS impairment (Fig.  2). NRTIs actually act as chain terminators because their incorporation into the growing chain of mtDNA does not allow the addition of endogenous nucleotides by the DNA polymerase γ.22, 30, 50 Therefore, the lack of a 3′-hydroxyl group is responsible not only for the antiretroviral activity of NRTIs, but also explains their mitochondrial toxicity. Zalcitabine is not used anymore because of its much higher toxicity than other NRTIs. Some NRTIs, in particular stavudine and didanosine, are suspected to worsen NAFLD in obese patients.6, 51 Although the mechanisms of NRTI-induced NAFLD aggravation are still unclear, it is likely that mitochondrial dysfunction could play a significant role.6, 51 Several studies suggested that the risk of stavudine-induced lactic acidosis could be higher in overweight or obese female patients.52, 53 Moreover, a high body mass index might increase the risk of fatal lactic acidosis.54 It is conceivable that stavudine treatment might have significantly exacerbated NAFLD-associated mitochondrial dysfunction in obese individuals, triggering severe TCA cycle defect and lactic acidosis. Unfortunately, these investigations about stavudine-induced lactic acidosis did not determine whether this antiretroviral drug also worsened fatty liver or NASH in these patients.

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5 Ethanol

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4.3 Tamoxifen Tamoxifen is a selective estrogen-receptor modulator used for the treatment of estrogen receptor-positive breast cancer. Tamoxifen-induced hepatotoxicity mostly includes different types of chronic liver lesions, including steatosis, steatohepatitis, fibrosis, cirrhosis, and HCC.36, 55 Investigations performed on isolated rodent liver mitochondria have shown that tamoxifen is able to impair OXPHOS and MRC activity in a similar manner than amiodarone (Fig. 2), with OXPHOS uncoupling at relatively low tamoxifen concentrations (50–100 μM) and inhibition of MRC activity at higher concentrations.56–58 In addition to its effect on OXPHOS, tamoxifen is also able to directly inhibit mtFAO (Fig. 2), in particular at the level of CPT1.58 Investigations of mice showed that chronic tamoxifen treatment (i.e., 28 days) induced progressive hepatic mtDNA depletion, possibly via an impairment of mitochondrial topoisomerase activity.58 In recent experiments performed in differentiated HepaRG cells treated for 14 days with tamoxifen, however, we failed to detect any mtDNA depletion, whereas treatment with the NRTI zalcitabine induced a strong reduction in mtDNA levels (unpublished results). Although all these mitochondrial effects most probably explain why tamoxifen is able to induce steatosis,12, 25, 58 some investigations suggested that increased lipogenesis could also be involved in the accumulation of liver triglycerides.59, 60 Several studies have reported that obesity increased the risk of tamoxifen hepatotoxicity as assessed by the presence of hepatic histological alterations or increased plasma transaminases.61–63 Two of these studies showed that obesity more specifically enhanced the risk of tamoxifen-induced steatohepatitis.62, 63 In a study reporting three cases of tamoxifen-induced steatohepatitis, all patients were overweight, or obese.64 The study from Saphner et  al.,63 however, suggested that tamoxifen did not worsen pre-existing NASH in a small subgroup of patients. Further investigations in larger series of obese patients undergoing serial liver biopsies would be needed to determine whether tamoxifen can actually worsen pre-existing NASH, or whether tamoxifen can induce steatohepatitis more frequently in patients with pre-­existing fatty liver. Nevertheless, whatever the clinical situation, it seems likely that tamoxifen-­induced mitochondrial dysfunction could be involved.

5 ETHANOL Even though low levels of ethanol can be found in the blood of nonalcoholic individuals, this molecule is mentioned in this chapter because its overconsumption is a major issue for public health. The abuse of alcoholic beverages can induce a large spectrum of liver lesions, including alcoholic foamy degeneration, alcoholic hepatitis, macrovacuolar steatosis, steatohepatitis, cirrhosis, and HCC.3, 27 A large body of evidence indicates that oxidative stress is a major mechanism whereby ethanol intoxication induces acute and chronic liver injury. Ethanol abuse is associated with ROS overproduction because of the induction of cytochrome P450 2E1 (CYP2E1) and MRC impairment.3, 65, 66 ROS can damage different cellular components, including unsaturated fatty acids, therefore generating highly reactive aldehydes such as malondialdehyde and 4-hydroxynonenal that play a major role in alcoholic liver diseases.3, 67 Ethanol intoxication also impairs ROS detoxification, in particular by reducing glutathione levels within the cytosol and mitochondria.27, 68 Moreover, ethanol metabolism generates

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a­ cetaldehyde and the hydroxyethyl radical, which are highly reactive metabolites able to alter many cellular components.3, 69 Overproduction of ROS and lipid peroxidation-derived aldehydes, as well as the generation of ethanol-derived toxic metabolites, can impair mitochondrial function and alter different mitochondrial components (Fig. 2). ROS and toxic metabolites produced after ethanol consumption could be generated directly within mitochondria because hepatic CYP2E1 is present at significant levels in these organelles.70, 71 Ethanol-induced impairment of MRC activity and OXPHOS can be induced by different mechanisms, including direct alterations of key components of the MRC such as COX and ATP synthase, impairment of mitochondrial protein synthesis and oxidative damage to the mitochondrial genome leading to different point mutations, large deletions, and reduced mtDNA copy number (Fig. 2).22, 23, 27, 72, 73 Ethanol intoxication also impairs mtFAO (Fig. 2) by different mechanisms, including reduced NAD+ availability secondary to ethanol metabolism by alcohol and aldehyde dehydrogenases, reduced activity of several mtFAO enzymes such as CPT1 and medium-chain acyl-CoA dehydrogenase (MCAD), and alteration of peroxisome proliferator-activated receptorα (PPARα) signaling pathway.27, 74–76 The latter effect seems dependent on oxidative stress and acetaldehyde generation.74, 76 mtFAO could also be impaired secondary to reduced MRC activity. Although impairment of mtFAO undoubtedly plays a major role in ethanol-induced steatosis,27, 73, 77 other mechanisms could also participate to fat accretion including increased de novo lipogenesis, higher esterification of fatty acids to triglycerides and reduced very-low density lipoprotein (VLDL) secretion.3, 27, 78 Strong evidence suggests that excess weight and obesity greatly increase the risk and the severity of alcoholic liver disease, in particular steatohepatitis.79–81 Although several mechanisms seem to be involved, the higher basal CYP2E1 activity associated with NAFLD could play a significant role by enhancing ethanol-induced oxidative stress, lipid peroxidation, and mitochondrial dysfunction.81–83 This, in turn, could explain stronger mitochondrial dysfunction and ATP shortage84 and secondary necroinflammation.83 CYP2E1-independent mechanisms, however, could also be involved because higher ethanol-induced liver injury can be observed in genetically obese fa/fa rats and ob/ob mice that do not present basal CYP2E1 induction.82, 85

6  OCCUPATIONAL AND ENVIRONMENTAL POLLUTANTS 6.1 Benzo[a]pyrene Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) found in crude oil, coal tar, tobacco, and many foods, especially grilled meats. B[a]P is listed as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). B[a]P is a potent activator of the aryl hydrocarbon receptor (AhR), which is involved in different cellular effects of this PAH including induction of different CYPs (CYP1A1/2 and CYP1B1) and glycolytic reprogramming.86, 87 Several CYP-generated oxygenated B[a]P metabolites are highly toxic for the cells, in particular by forming covalent DNA adducts.86, 88 B[a]P has been reported to induce lipid deposition in hepatocytes in different experimental models.89–91 This effect seems to be linked to an increase in de novo lipogenesis,90, 91 ­possibly

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via an activation of AhR by B[a]P.90 Recent investigations in our laboratory, however, showed that a 14-day treatment with B[a]P reduced triglyceride content in human HepaRG cells when these cells were overloaded or not with a mixture of stearic and oleic acids.92 These effects were associated with higher mtFAO.92 Previous investigations in chick embryos also reported that B[a]P was able to increase mtFAO, possibly via the generation of an oxygenated metabolite.93 Therefore, the exact effects of B[a]P on hepatic lipid metabolism require further investigations. Our experiments also showed that B[a]P reduced the activity of the MRC complexes I and IV in lipid-overloaded HepaRG cells but not in nonsteatotic cells.92 These effects in steatotic HepaRG cells were associated with ROS overproduction and a pro-inflammatory state with higher mRNA expression of interleukin-1β (IL-1β) and its receptor IL1R1, as well as increased IL-6 secretion (92 and unpublished data). MRC impairment, ROS overproduction, and higher interleukin expression induced by the 14-day exposure of B[a]P were aggravated by ethanol co-exposure. Therefore, B[a]P might favor the pathological progression of liver steatosis to a steatohepatitis-like state, possibly by promoting MRC impairment, ROS overproduction, and inflammation.92 We surmise that B[a]P-induced mitochondrial ROS overproduction might be particularly favored by the concomitant increase in the mtFAO flux and lower MRC activity.92 The precise mechanisms whereby B[a]P alters MRC activity are still unknown. Some investigations suggested the involvement of CYP-derived toxic metabolites and ROS.94 These deleterious molecules could target different MRC complexes directly but also could damage the mitochondrial genome.94, 95 Our recent investigations in HepaRG cells, however, suggested that B[a]P-induced impairment of MRC activity could be unrelated to the formation of CYPderived metabolites, suggesting that the mere binding of B[a]P to AhR could alter MRC activity.92 AhR is translocated to mitochondria and can interact with mitochondrial proteins such as ATP5α1, a subunit of ATP synthase.96, 97 These investigations, however, did not determine whether B[a]P-induced MRC impairment was secondary to the interaction of this PAH with the mitochondrial AhR.

6.2  Carbon Tetrachloride Carbon tetrachloride (CCl4), also referred to as tetrachloromethane, is a chlorinated hydrocarbon used as a solvent, refrigerant, fire extinguisher, and dry-cleaning agent. The production and use of this compound have decreased significantly during the past decades because of the occurrence of numerous cases of multiorgan toxicity in industrial workers and the adoption of the Montreal Protocol banning chlorofluorocarbon gases. In addition to occupational exposure, CCl4 can be found in the ambient air and groundwater supplies.98 CCl4-induced hepatotoxicity in humans and animals includes acute liver injury with necrosis and steatosis as prominent pathologic features, as well as chronic liver injury including steatosis, steatohepatitis, cirrhosis, and HCC.1, 98, 99 Repeated administration of CCl4 in rodents is used as a classic experimental model of extensive fibrosis and cirrhosis. Similar to ethanol intoxication, the primary mechanisms involved in CCl4-induced liver injury include CYP2E1-mediated generation of highly reactive metabolites that covalently bind to cellular macromolecules, reduce GSH levels, induce ROS overproduction, and promote lipid peroxidation.98–100 In turn, these deleterious events induce mitochondrial dysfunction, including

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reduced activity of different MRC complexes, OXPHOS impairment, and mtFAO inhibition (Fig. 2).33, 100–102 CCl4 can also induce oxidative damage to the mitochondrial genome, leading to mtDNA strand breaks and reduced mtDNA copy number (Fig. 2).33, 102 CCl4-induced mitochondrial dysfunction seems to play a significant role, not only in the occurrence of steatosis and necroinflammation, but also in fibrosis progression.33, 103, 104 In addition to mitochondrial dysfunction, CCl4 could also favor hepatic triglyceride accumulation via a reduction of VLDL secretion.1, 100 Several experimental studies carried out in rodents showed that NAFLD and type 2 diabetes can sensitize to CCl4-induced acute hepatotoxicity.105–107 Unfortunately, these studies did not investigate the exact role of mitochondrial dysfunction.

6.3 Organochlorines 6.3.1  Polychlorinated Biphenyls (PCBs) Polychlorinated biphenyls (PCBs) are organic chlorine compounds that have wide industrial use as coolants and insulating fluids, plasticizers in paints and cements, wax extenders, and flame retardants. There are more than 200 PCBs, which can be classified as dioxin-like and nondioxin-like compounds based on the similarity of their toxicity profile to 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). Examples of dioxin-like PCBs include 3,3′,4,4′-­ tetrachlorobiphenyl (PCB 77) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126). PCBs were also commercially produced as mixtures, which are referred to as Aroclors. Because of PCBs’ environmental toxicity and classification as persistent organic pollutants (POPs), their production was banned by the Stockholm Convention on POPs in 2001. IARC classified PCBs as Group 1 carcinogens in humans. Even though PCB levels are decreasing in the environment, PCB exposure still remains a major toxicological issue.98 In humans, exposure to PCBs is suspected to contribute to obesity, diabetes, hypertension, and NAFLD.98, 108, 109 The ability of some PCBs to induce steatosis and steatohepatitis has been confirmed in rodents.1, 98, 110 The mechanisms whereby PCBs can induce steatosis and steatohepatitis are still poorly understood and most probably differ between compounds. For example, PCBs can interact differentially with several nuclear receptors that have complex effects on lipid metabolism, such as AhR, constitutive androstane receptor (CAR), and pregnane xenobiotic receptor (PXR).111 AhR activation can induce hepatic steatosis by different mechanisms, as discussed later for TCDD. Several in vitro studies also showed that several PCBs can directly impair mitochondrial function, in particular at the level of different MRC complexes, OXPHOS, and mtFAO.112–115 These deleterious effects, however, were significant at rather high concentrations of PCBs (>10–20 μM), and therefore it is unclear whether mitochondrial dysfunction might play a role in PCB-induced steatosis in the context of environmental exposure. Experimental investigations reported that some PCBs, such as PCB 77 and PCB 153, were able to worsen fatty liver in mice fed a high-fat diet (HFD).116–118 PCB 153-induced aggravation of fatty liver was associated with a profound decrease in hepatic PPARα expression that was accompanied by a strong reduction of CPT1 and CPT2 expression.118 The mtFAO pathway, however, was not directly investigated in this study. Previous investigations showed that another PCB, namely PCB 77, was able to potently reduce PPARα mRNA expression at very low concentrations (i.e., 1 nM).119 Lastly, worsening of fatty liver was not observed in HFD obese mice exposed to Aroclor 1260, indicating that such hepatic effect is not observed

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with all PCBs.120 Nonetheless, Aroclor 1260 caused necroinflammation in HFD obese mice, suggesting that this PCB mixture could favor the progression of fatty liver to NASH.120 6.3.2 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) TCDD (also referred to as dioxin) is an organochloride usually formed as a byproduct during organic synthesis and waste combustion. For instance, TCDD was a contaminant in Agent Orange, an herbicide used during the Vietnam War, that contained an equal mixture of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). This persistent contaminant was also massively released into the environment following different industrial accidents, including the Seveso disaster in Italy in 1976.121 Acute intoxication by TCDD leads to different adverse effects, including severe skin lesions such as chloracne, dehydration, weight loss, peripheral neuropathy, and hepatotoxicity.121, 122 Delayed or long-term intoxication by TCDD is also suspected to contribute to the occurrence of dyslipidemia, atherosclerosis, hypertension, diabetes, chronic liver disease, and different types of cancers.121–123 Accordingly, TCDD is listed as a Group 1 carcinogen by IARC. In humans, TCDD-induced hepatotoxicity includes increased plasma transaminases (reflecting hepatic cytolysis), steatosis, fibrosis, and inflammation, suggesting the occurrence of steatohepatitis in some individuals.121, 122 Steatosis is also consistently observed in rodents intoxicated by TCDD.124–128 Notably, necroinflammation and fibrosis were observed in some of these investigations and others,124, 125, 128, 129 confirming that TCDD is able to induce steatohepatitis. Mechanistic studies suggested that TCDD-induced steatosis could be induced by different mechanisms including increased hepatic fatty acid uptake, reduced VLDL secretion and decreased mtFAO.124, 126, 128, 130 Some of these detrimental effects could be linked to AhR activation,126, 131, 132 although other mechanisms cannot be excluded. TCDD also impairs the activity of different MRC complexes, which could participate to higher mitochondrial ROS production.96, 133, 134 Interestingly, TCDD-induced mitochondrial ROS overproduction seems to be secondary to AhR activation.135 Finally, a recent study reported that TCDD promotes liver fibrosis development in HFD obese mice, but the possible involvement of mitochondrial dysfunction was not addressed in this study.136

7  CONCLUDING REMARKS AND PERSPECTIVES Strong evidence exists that many xenobiotics are able to induce steatosis and steatohepatitis. Although steatosis is a benign condition, steatohepatitis is a major issue for the affected patients because the lesion can progress to cirrhosis and eventually to HCC. Therefore, it is important to know the full spectrum of xenobiotics able to induce steatosis and steatohepatitis, as well as the populations at risk. Although prospective clinical investigations and broad epidemiologic studies could help in this task, it must be stressed that the clear identification of the involved compounds is difficult in particular in patients with polymedication, or in individuals exposed to various environmental pollutants. Experimental investigations in vitro and in vivo are important to ascertain whether a given molecule is able to induce steatosis or steatohepatitis. Regarding in vitro experiments, several investigations showed that the HepaRG cell line is a valuable model to investigate steatosis induced by drugs and ­environmental

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­ ollutants but also by fatty acid overload.92, 137–141 HepaRG cells express the main xenobioticp metabolizing enzymes (XMEs), such as CYPs and UDP-glucuronosyltransferases (UGTs), as well as the nuclear receptors that control XME expression.141–143 Recent investigations showed that mitochondrial function in HepaRG cells is close to primary human hepatocytes, in contrast to HepG2 and Huh7 cells.144, 145 This is a key feature because xenobiotics can induce steatosis and steatohepatitis by impairing mitochondrial function. In contrast to obesity-associated fatty liver, the mechanisms whereby xenobiotics can induce steatosis are much less known. Although mitochondrial dysfunction is deemed to play a key role, at least for some xenobiotics, other mechanisms exist such as activation of hepatic de novo lipogenesis and impairment of VLDL secretion, as previously mentioned. In addition, some xenobiotics, including drugs, ethanol, and some environmental pollutants, could favor hepatic steatosis secondary to their deleterious effects on adipose tissue.25, 146, 147 Therefore, although cellular models are valuable to study xenobiotic-induced steatosis, in vivo investigations in rodents could help to disclose indirect toxicity on the liver. In addition, investigations in HFD or genetically obese rodents could determine whether a given molecule can aggravate fatty liver, or can favor the progression of fatty liver to steatohepatitis.85, 116–118, 148 Finally, epidemiological studies might also help to tackle this issue, although this approach could be difficult for populations that are exposed to many environmental pollutants.

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IV.  FACTORS THAT MAY TRIGGER OR AGGRAVATE THE PATHOLOGIES